PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is a dangerous human pathogen due to its large repertoire of virulence factors, which allow this bacterium to infect any part of the human body. Our long-term goal is to understand the mechanisms by which S. aureus modulates virulence factor expression and activity to cause disease. This includes uncovering novel pathways contributing to virulence factor production and responses to environmental changes. During an unbiased transposon library screen, mutants within a two-gene operon were found to lack production of a key protease and to have less pigment. The particular protease, aureolysin, is essential to modulating the extracellular proteome and cleavage of host proteins while the pigment, staphyloxanthin, has been demonstrated to play a role in oxidative stress resistance. While annotated as encoding hypothetical proteins, further analysis indicates that the genes identified in the transposon study encode a truncated hemoglobin (YjbI) and a thioredoxin-like thiol/disulfide oxidoreductase (YjbH). The YjbI designation comes from Bacillus, where the structure, but not function, of a similar protein has been solved. Limited studies on YjbH demonstrate that it is involved in the stability of the Spx regulatory protein, also primarily in Bacillus. To date, neither has been shown to play a role in virulence factor regulation or pathogenesis. Truncated hemoglobins remain poorly understood in bacteria, but have been implicated in oxygen sensing and the response to oxidative and nitrosative stress. Similarly, thioredoxin-related proteins also play a role in oxidative and nitrosative stress. Our preliminary data demonstrate that YjbIH contribute to protease regulation, pigmentation, and resistance to oxidative and nitrosative stress. Further analyses revealed that deletion of these genes led to altered virulence in a murine model of infection. Based on our preliminary data, we hypothesize that YjbI and/or YjbH of S. aureus modulates the stress response, leading to altered virulence factor expression and host colonization. To test this, two aims are proposed. Aim 1 examines key mutants in assays to identify the regulatory networks influenced by YjbIH and their impact on aureolysin, pigmentation, and the oxidative and nitrosative stress response. Unbiased assays will determine global transcriptomic changes in the yjbIH strain and will identify binding partners for YjbI and YjbH. Aim 2 builds upon our existing in vivo data and additional mutants to uncover the virulence factors necessary for enhanced colonization of the yjbIH mutant. Completion of these studies will provide insight into virulence factor regulation in this important pathogen. Additionally, it will shed light on the function of these ubiquitous, yet understudied proteins. In addition to the research plan, a training plan has been put in place for Dr. Austin, who has extensive teaching experience. As highlighted in the application, the primary goal is to prepare her for becoming an independent investigator. The plan utilizes the strengths of our interdisciplinary department, collaborators, and active Office of Postdoctoral Affairs to train her in research program development, mentoring, and laboratory management.